Er clone 1d5

Manufactured by Agilent Technologies

Sourced in Denmark, United Kingdom

The ER (clone 1D5) is a laboratory equipment product from Agilent Technologies. It is designed to perform specific functions in research and laboratory settings. The core function of this product is to [description not available].

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Mouse monoclonal anti-ER (clone 1D5, Anti-ER (clone 1D5 ER (1D5, Clone 1D5

8 protocols using er clone 1d5

Immunohistochemical Evaluation of Tumors

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Tumor grading, tumor typing and immunohistochemistry (ER, PR, Ki-67) was performed on the pretreatment core biopsies on all patients. Pathological complete response (pCR) was determined on tumor resection specimens after completion of neoadjuvant chemotherapy, and was defined as no evidence of residual invasive and ductal disease in the breast and lymph nodes (ypT0,ypN0).
Immunohistochemistry was performed according to previously standardized protocols on an automated IHC platform (Dako Techmate 500) with citrate buffer for antigen retrieval [23 (link)] and observing the ASCO/CAP guidelines for immunohistochemistry [7 (link)]. The following primary antibodies and corresponding dilutions were used (DakoCytomation, Glostrup, Denmark): ER (clone 1D5, 1:100), PR (clone PgR636, 1:100) and Ki-67 (MIB-1, 1:200). Slides were assessed by quantitative image analysis (qIHC) using the Aperio Image Analysis toolbox (Leica Biosystems, Nussloch, Germany). Staining intensity and percentage of positive nuclei were recorded after manually segmenting tumor from adjacent stroma. Tumors with ER/PR Remmele scores greater than 3 or positive nuclei greater than 1% were considered hormone receptor positive.

Sinn H.P., Schneeweiss A., Keller M., Schlombs K., Laible M., Seitz J., Lakis S., Veltrup E., Altevogt P., Eidt S., Wirtz R.M, & Marmé F. (2017). Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer. BMC Cancer, 17, 124.

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Immunohistochemical Evaluation of ER Expression

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Three to four micrometer slides from selected formalin-fixed, paraffin-embedded tissues were prepared for envision IHC staining.
After preparation of 4 µm thin slides, sections were placed on the poly-l-lysine slides, and then they were deparaffinized and dried in an oven at 60°C for 60 min. After rehydrate, their antigens were retrieved by boiling them in Tris-buffered saline by microwave heat-induced epitope retrieval method. After inactivation of endogenous catalase by using 3% hydrogen peroxide, the slides were incubated with antibodies to ER clone 1D5 (Dako, Carpinteria, CA) for 1 h and secondary antibody for 30 min, and then antibodies was localized and made apparent by streptavidin-biotin method and diaminobenzidine as chromogen. Stained slide was considered as positive if there was nuclear staining: 1 (upto 10% of cells positive), 2 (11–50% positive), and 3 (>50% positive).

Rajabi P., Bagheri M, & Hani M. (2017). Expression of Estrogen Receptor Alpha in Malignant Melanoma. Advanced Biomedical Research, 6, 14.

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Immunohistochemical Analysis of Breast Cancer

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All patients underwent a CNB before NAC, and an operation consisting of mastectomy or conserving surgery with axillary lymph node dissection after NAC at Osaka City University. Tissues from each patient were fixed in buffered formalin and embedded in paraffin. Serial tissue sections of 4 μm thickness were stained with hematoxylin-eosin and used for immunohistochemical staining. Expressions of CA9, estrogen receptor (ER), progesterone receptor (PgR), and HER2 were assessed by immunohistochemistry. After the paraffin sections were deparaffinized, they were heated for 20 min at 105°C by autoclave in Target Retrieval Solution (Dako, Carpinteria, CA). After blocking with 10% goat serum, the slides were incubated with the primary monoclonal antibodies against each of CA9 (clone M75, 1:1000; Novus Biologicals), ER (clone 1D5, dilution 1:80; Dako, Cambridge, UK), PgR (clone PgR636, dilution 1:100; Dako), and HER2 (Hercep Test, Dako) overnight at 4 °C. Peroxidase was introduced using a streptavidin conjugate and then peroxidase reactivity was visualized using a DAB solution, followed by counterstaining with haematoxylin.

Aomatsu N., Yashiro M., Kashiwagi S., Kawajiri H., Takashima T., Ohsawa M., Wakasa K, & Hirakawa K. (2014). Carbonic anhydrase 9 is associated with chemosensitivity and prognosis in breast cancer patients treated with taxane and anthracycline. BMC Cancer, 14, 400.

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Immunohistochemical Assay for Breast Cancer Subtypes

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An immunohistochemical assay was used to test for expression of the estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67. Immunohistochemistry was performed for ER (clone 1D5), PR (PgR636), HER2, and Ki-67 (clone MIB-1), all of which were from Dako (Glostrup, Denmark), as a routine diagnostic procedure. The cut-off values for ER and PR positivity were defined as ≥1% tumor cells with nuclear staining. Immunohistochemical staining for HER2 was scored according to standard criteria as 0, 1+, 2+, or 3+.15 (link) Scores of 0 and 1+ were considered negative, and 3+ was considered to be HER2-positive. When a score of 2+ was found, additional fluorescent in situ hybridization testing was performed to establish the HER2 gene amplification status. A positive result was defined as an HER2 gene/chromosome 17 ratio of ≥2.0. A positive Ki-67 finding was defined as ≥14%, and a negative finding was defined as <14%.16 (link) The subtype was proposed to separate the luminal A (ER+/PR+, HER2−, Ki-67 <14%), luminal B/HER2− (ER+/PR+, HER2−, Ki-67 ≥14%), luminal B/HER2+ (ER+/PR+, HER2+), HER2-enriched (ER−, PR−, HER2+) and triple-negative (ER−, PR−, HER2−) subtypes.

Zhao Y., Dong X., Li R., Ma X., Song J., Li Y, & Zhang D. (2015). Evaluation of the pathological response and prognosis following neoadjuvant chemotherapy in molecular subtypes of breast cancer. OncoTargets and therapy, 8, 1511-1521.

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Estrogen Receptor Immunohistochemistry in Tumors

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Immunohistochemical staining was performed for the characteristic areas of tumors (1–2 paraffin blocks per case) from microscopically selected samples (regions), based on standard (hematoxylin and eosin) H&E staining. Tissue from the paraffin molds was cut into 4-µm-thick sections and placed on Superfrost glass slides. Antigen retrieval of deparaffinized and rehydrated samples was done in citric acid buffer (pH 6.0) in a microwave oven for 20 min. After cooling to room temperature, the blockage of endogenous peroxidase was performed using 3% (w/w) hydrogen peroxide. After sample washing (PBS, pH 7.4), primary estrogen (Monoclonal Mouse Anti-Human Estrogen Receptor α (ER); Clone 1D5; Code N1575, Ready-to-use; Dako, Glostrup Denmark) antibody was applied for 40 min at room temperature in a moist chamber. Visualization was achieved by incubation of slides with Dako LSAB2 System-HRP (Code K0673, 15 mL) and diaminobenzidine (DAB), followed by washing and counterstaining with Mayer’s hematoxylin.

Ilić I.R., Stojanović N.M., Radulović N.S., Živković V.V., Randjelović P.J., Petrović A.S., Božić M, & Ilić R.S. (2019). The Quantitative ER Immunohistochemical Analysis in Breast Cancer: Detecting the 3 + 0, 4 + 0, and 5 + 0 Allred Score Cases. Medicina, 55(8), 461.

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Comprehensive IHC Assay for FFPE Specimens

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An immunohistochemistry (IHC) assay of formalin-fixed and paraffin-embedded (FFPE) specimens was performed using Dako’s EnVision System (Glostrup, Denmark). This is a 2-step method in which the application of the primary antibody is followed by a polymeric conjugate consisting of many secondary antibodies bound directly to a dextran backbone, including ER (clone1D5, DAKO), PR (Clone pgR 1294, DAKO), P53 (Clone DO-7, DAKO) and HER2 (VENTANA anti-HER2/neu(4B5), Roche Diagnostics GmbH). Procedures were provided by the manufacturers and completed on a Roche automated IHC instrument (USA). The antibodies and IHC procedures used in this study were part of a standard IHC panel for a pathology laboratory.

Lian J., Xu E.W., Xi Y.F., Wang H.W., Bu P., Wang J.F, & Wang L.X. (2020). Clinical-Pathologic Analysis of Breast Cancer With PIK3CA Mutations in Chinese Women. Technology in Cancer Research & Treatment, 19, 1533033820950832.

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IHC and 21-gene Recurrence Score Assay

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Pathological and IHC analysis was performed at the Department of Pathology, Ruijin Hospital. IHC staining was performed on 4-μm-thick FFPE tissue section with the following antibodies: ER (clone 1D5; 1:100; rabbit monoclonal; Dako), HER2 (clone 4B5, rabbit monoclonal; 1:100; Roche). ER positivity was defined as nuclear staining in ≥1% of tumor cells. HER2 was considered negative with 0–1 by IHC or negative by FISH. The 21-gene RS assay was performed on FFPE tissues as described in our previous study [13 (link)]. Total RNA was extracted from three 10-μm unstained sections using the RNeasy FFPE RNA kit (Qiagen, Germany) after identifying the absence of DNA contamination. Gene-specific reverse transcription was performed on the Omniscript RT kit (Qiagen, Germany) followed by standardized qPCR with Premix Ex TaqTM (Takara Bio, Inc.) in Applied Biosystems 7500 Real-Time PCR system (Foster City, CA). The expression levels of 16 cancer-related genes were measured in triplicate and normalized by 5 reference genes. The RS, ranging from 0 to 100, was then calculated by using a specific algorithm [3 (link)].

Yu J., Lin C., Huang J., Hong J., Gao W., Zhu S., Lin L., Chen X., Huang O., He J., Zhu L., Chen W., Li Y., Wu J, & Shen K. (2021). Efficacy of adjuvant chemotherapy stratified by age and the 21-gene recurrence score in estrogen receptor-positive breast cancer. BMC Cancer, 21, 707.

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Immunohistochemical Analysis of Breast Cancer

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All patients underwent a core needle biopsy prior to NAC and curative surgery involving mastectomy or conservative surgery with axillary lymph node dissection after NAC. Immunohistochemical studies were performed as described previously.17 (link) Primary monoclonal antibodies directed against ER (clone 1D5, dilution 1:80; Dako), PgR (clone PgR636, dilution 1:100; Dako), HER2 (HercepTest; Dako), Ki-67 (clone MIB-1, dilution 1:00; Dako), CD8 (clone C8/144B, dilution 1:100; Dako) and FOXP3 (clone 236A/E7, dilution 1:100; Abcam, Cambridge, UK) were used.

Goto W., Kashiwagi S., Asano Y., Takada K., Takahashi K., Hatano T., Takashima T., Tomita S., Motomura H., Ohsawa M., Hirakawa K, & Ohira M. (2018). Predictive value of improvement in the immune tumour microenvironment in patients with breast cancer treated with neoadjuvant chemotherapy. ESMO Open, 3(6), e000305.

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